CN110772635B - Bionic nano vaccine coated by influenza virus corpuscle and preparation method thereof - Google Patents

Bionic nano vaccine coated by influenza virus corpuscle and preparation method thereof Download PDF

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CN110772635B
CN110772635B CN201911097481.1A CN201911097481A CN110772635B CN 110772635 B CN110772635 B CN 110772635B CN 201911097481 A CN201911097481 A CN 201911097481A CN 110772635 B CN110772635 B CN 110772635B
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influenza virus
influenza
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CN110772635A (en
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陈刚
周昕
李真真
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Yangzhou University
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • A61K9/141—Intimate drug-carrier mixtures characterised by the carrier, e.g. ordered mixtures, adsorbates, solid solutions, eutectica, co-dried, co-solubilised, co-kneaded, co-milled, co-ground products, co-precipitates, co-evaporates, co-extrudates, co-melts; Drug nanoparticles with adsorbed surface modifiers
    • A61K9/143—Intimate drug-carrier mixtures characterised by the carrier, e.g. ordered mixtures, adsorbates, solid solutions, eutectica, co-dried, co-solubilised, co-kneaded, co-milled, co-ground products, co-precipitates, co-evaporates, co-extrudates, co-melts; Drug nanoparticles with adsorbed surface modifiers with inorganic compounds
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A61P31/16—Antivirals for RNA viruses for influenza or rhinoviruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/53—DNA (RNA) vaccination
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
    • C12N2760/00011—Details
    • C12N2760/16011—Orthomyxoviridae
    • C12N2760/16111—Influenzavirus A, i.e. influenza A virus
    • C12N2760/16134—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein

Abstract

The invention discloses a bionic nano vaccine coated by influenza virus bodies (VI) and a preparation method thereof, wherein the bionic nano vaccine comprises the influenza virus bodies, small-particle-size fluorinated particles and a DNA vaccine, the bionic nano vaccine is a core-shell structure nano system, VI is a lipid vesicle containing influenza virus envelope protein, the inner core of the nano system is the small-particle-size fluorinated particles loaded with the DNA vaccine, and the influenza virus bodies are coated on the surface of the inner core. The virosome of the invention has the receptor binding activity, the lysosome membrane fusion activity and the antigen activity of influenza virus; the small size of the fluorinated inner core can deliver DNA into the core and promote protein expression. The nano system can realize the joint loading of the protein vaccine and the DNA vaccine and the site-specific delivery of each component, and finally obtains a synergistic effect to improve the immune effect. The invention belongs to the field of pharmaceutical preparations and the technical field of biological medicines, can effectively prevent influenza virus infection, and has high clinical application value.

Description

Bionic nano vaccine coated by influenza virus corpuscle and preparation method thereof
Technical Field
The invention relates to the field of pharmaceutical preparations and the technical field of biological medicines, in particular to a bionic nano vaccine coated by influenza virus corpuscles and a preparation method thereof.
Background
Seasonal influenza virus spread poses a great threat to global public health safety, and seriously affects human health and economic development. Vaccination remains the primary means of controlling and coping with influenza virus, and currently approved influenza vaccines are mainly inactivated vaccines (e.g., split vaccines) and live attenuated vaccines. However, influenza vaccines based on virus strain modification and inactivation still have more potential safety hazards in the using process, and mainly include adverse reactions such as back mutation, allergy, autoimmunity and the like of pathogens. In contrast, DNA vaccines and protein vaccines have the advantages of good safety, high purity, strong specificity and the like, but simple DNA and protein are difficult to be taken up by Antigen Presenting Cells (APC), and only short-term immunity or humoral immunity driven by Th2 cells can be induced, so that the application is greatly limited. In order to enhance the immune effect of DNA and protein vaccines, experts from the fields of biology, medicine and materials science and the like are more and more concerned about the application of nanotechnology in the vaccine field.
VI is derived from the host cell membrane during virus propagation and contains the virus' own glycoproteins. Glycoproteins on the surface of influenza virions include HA and NA, where HA binds to sialic acid receptors of host cells during viral infection and mediates endocytosis of the virus. The study shows that VI prepared in vitro is a unilamellar vesicle which is spherical and HAs an average particle size of less than 200nm, and compared with the liposome prepared artificially, the VI contains functional glycoprotein HA and NA but does not contain virus genetic material. VI prepared in vitro retains the receptor binding activity, the lysosome membrane fusion activity and the antigenicity of the influenza virus. Wherein, the receptor binding activity is beneficial to the APC to take up the nano-vaccine, the lysosome membrane fusion activity is beneficial to the lysosome escape of the inner core of the nano-particle, and the antigen activity can be directly used as the vaccine. Therefore, VI is a very potential carrier system and vaccine material.
DNA vaccines must enter the nucleus and be transcribed and translated to induce a corresponding immune response, but various cellular barriers limit the nuclear delivery of foreign DNA, mainly including the cell membrane barrier, inclusion body/lysosome barrier, and nuclear membrane barrier. Currently, various technical means are available to promote transmembrane transport of DNA, most commonly plasmid DNA (pDNA) is complexed with cationic liposomes, and the complex then interacts with negatively charged glycoproteins on the cell membrane to promote nonspecific endocytosis of the complex. For the inclusion body/lysosome barrier, lysosomes are burst primarily by increasing the osmotic pressure of the inclusion body/lysosome to achieve inclusion body/lysosome escape. The nuclear membrane barrier has long been a major factor that plagues the transcription of DNA into the nucleus. The currently common strategy is to modify the plasmid with a Nuclear Localization Sequence (NLS) peptide to increase the probability of entering the nucleus. However, the expression efficiency of pDNA modified with NLS is not high, and furthermore, charge interaction between anionic DNA and cationic NLS will bury NLS in DNA, limiting NLS function. Research shows that the nuclear pore size of the cell nucleus is 20-70 nm, and particles smaller than 50nm can enter the cell nucleus. In addition, the fluoridation modification can effectively improve the transfection efficiency and in vivo and in vitro stability of the cationic polymer, can effectively increase the nuclear distribution of the gene, and is beneficial to promoting the transcription expression of the pDNA.
Disclosure of Invention
The invention aims to: in order to achieve the purpose, the technical problem to be solved by the invention is to provide a bionic nano vaccine coated by influenza virus corpuscles.
The invention also aims to solve the technical problem of providing a preparation method of the bionic nano vaccine coated by the influenza virosome.
The technical scheme is as follows: in order to solve the technical problems, the invention provides a bionic nano vaccine coated by an influenza virosome, which comprises an influenza Virosome (VI), small-particle-size fluorinated particles and a DNA vaccine.
The bionic nano vaccine is a core-shell structure nano system, the VI is a lipid vesicle containing influenza virus envelope protein, the inner core of the nano system is small-particle-size fluorinated particles for loading DNA vaccine, and the influenza virus corpuscle is wrapped on the surface of the inner core.
Wherein the influenza virus corpuscles are derived from influenza viruses, and the influenza viruses include, but are not limited to, subtypes of H1N1, H3N2, H5N1, H7N9, H9N2 and the like.
The small-particle-size fluorinated particles are nanostructures formed by wrapping gold particles with fluorinated modified cationic polymers.
Wherein the fluorinated modified cationic polymer is a gene vector, including but not limited to polyamines such as linear or branched Polyethyleneimines (PEI); polyamide-amines (PAA), such as dendrimers PAMAM, hyperbranched polyamide-amines (hpaam); polymethacrylates such as dimethylaminoethyl methacrylate (pDMAEMA); polyamino acids (polyaminoacids) such as Polylysine (PLL); polyesters (polyesters), such as linear or branched poly (. Beta. -aminoesters) (PBAE); natural polysaccharides (polysaccharides), such as chitosan, etc.
Wherein the particle size of the gold particles is 10-30 nm.
The invention also discloses a preparation method of the bionic nano vaccine coated by the influenza virosomes, which comprises the following steps:
1) VI preparation: adding 1, 2-dihexanoyl lecithin into influenza virus, performing ice bath, performing ultracentrifugation, collecting supernatant, dialyzing the supernatant in HBS buffer solution, and removing 1, 2-dihexanoyl lecithin to obtain recombinant VI;
2) Fluorinated modified cationic polymer synthesis: reacting according to the molar ratio of amino, heptafluorobutyric anhydride and triethylamine in the cationic polymer of 1:3:1.2, wherein the reaction medium is methanol, stirring at room temperature, dialyzing the reaction product in deionized water with pH of 3-4 after the reaction is finished, and freeze-drying to obtain the fluorinated modified cationic polymer;
3) Preparation of FAu: slowly dripping gold particles with the particle size of 10-30 nm into the fluorinated modified cationic polymer, slowly shaking lightly, centrifuging, and collecting precipitates to obtain fluorinated particles FAu with small particle size;
4) Preparation of DNA vaccine-loaded FAu: mixing the FAu obtained in the step 3) with the DNA vaccine, and slowly shaking the mixture to obtain small-particle-size fluorinated particles loaded with the DNA vaccine, wherein the mass ratio of the cationic polymer to the DNA is 1.5-3;
5) Preparation of ARV: and (3) mixing the VI recombined in the step 1) with the fluorinated particles with small particle sizes in the step 4), extruding back and forth by using an extruder, and centrifuging and collecting to obtain the ARV.
Wherein, the ultracentrifugation conditions of the step 1) are 4 ℃,100, 000 Xg and 1.5h.
Wherein the HBS buffer solution in the step 1) is a HEPES buffer solution containing 0.15 MNaCl.
Wherein the concentration of the fluorinated modified cationic polymer in the step 3) is 5-10 mg/mL.
Wherein, the DNA vaccine of the step 4) includes, but is not limited to, a full-length DNA sequence or a partial DNA sequence of Nucleoprotein (NP), matrix protein (M1), membrane protein (M2), hemagglutinin (HA) or Neuraminidase (NA) of influenza virus.
Wherein the size of the filter membrane hole of the extruder in the step 5) is 50-100 nm.
Has the advantages that: compared with the prior art, the invention has the advantages that: simple DNA and protein vaccines are difficult to effectively take by APC, and the induced immune response is weak. In addition, due to the differences of the properties of different active substances, the nano system needs to realize the common loading of all components and the effective release of different target sites, which is always a difficulty in the research of preparations. The nano vaccine disclosed by the invention is specifically combined with a sialic acid receptor on the surface of APC (APC) through HA (hyaluronic acid) on the surface, and mediates the endocytosis of particles into an endosome/lysosome; VI, fusing with a lysosome membrane to release the nano particles carrying the DNA vaccine to cytoplasm; the nano particles escaping from lysosome enter the cell nucleus for transcription and expression under the advantages of fluorination and small particle size of the particles. The nano vaccine simulates a virus replication mechanism, realizes site-specific sequential delivery of protein and DNA vaccines, and finally obtains a synergistic effect to improve the immune effect. The nano vaccine prepared by the invention has simple and controllable process, low production cost and good repeatability, and is suitable for large-scale production; the carrier has good stability and high safety. The vaccine can effectively reduce the infection risk of the influenza virus and can effectively control the infection caused by the influenza virus.
Drawings
FIG. 1 is a schematic diagram of the preparation principle of an influenza virosome-coated biomimetic nano vaccine of the present invention;
figure 2, biomimetic nano-vaccine characterization of influenza virosome-coated of the present invention;
a: the particle size of the bionic nano vaccine coated by the small influenza virus bodies is small;
b: the influenza virus corpuscle coating bionic nano vaccine has potential distribution;
c: a transmission electron microscope image of the bionic nano vaccine coated by the influenza virosome;
FIG. 3: the bionic nano vaccine film coated by the influenza virus corpuscle is fused and transmits plasmid DNA into a nucleus;
a: preparation of ARV and VI according to example 1 of the invention incubation with erythrocytes respectively, OD 540 The curve of the ultraviolet absorption with the change in pH;
b: the bionic nano vaccine coated by the influenza virus corpuscle is subjected to membrane fusion experiments at pH5.5 and pH7.4 respectively;
c: ARV' without fluorinated modified cationic polymer and ARV delivery plasmid DNA prepared according to the invention into the nucleus;
FIG. 4: the bionic nano vaccine coated by the influenza virus corpuscle has cellular immunity and humoral immunity response;
a: the bionic nano vaccine group coated by influenza virus corpuscle is ARV group: injecting nano vaccine subcutaneously in tail base part of 1 week and 3 weeks, separating spleen at 4 weeks, preparing single cell suspension, incubating with CD3 and CD8 antibody, and detecting CD8 in mouse body by flow cytometry + The amount of T cells; a salt group: injecting normal saline subcutaneously at tail base of 1 week and 3 weeks, and the rest is ARV group; group VI: subcutaneous injections of VI at the base of the tail at weeks 1 and 3, the remainder being in the ARV group; pFAU group: pFAu was injected subcutaneously at the base of the tail at weeks 1 and 3, and the rest were in the ARV group.
B: taking spleen single cell suspension, incubating with CD3 and CD4 antibody, detecting CD4 in mice by flow cytometry + The amount of T cells;
c: after immunization of mice, serum samples were collected at week 4 and IgG titers were determined;
FIG. 5: the bionic nano vaccine coated by the influenza virosome has a protective effect on virus infection;
a: influenza virusThe small-body coated biomimetic nano vaccine group is ARV group: injecting nanometer vaccine subcutaneously in tail base part of 1 week and 3 weeks, and attacking in 5 weeks, wherein each mouse is infected with 1 × 10 4 CFU PR8 virus, continuously recording the weight of the mice for 14 days; a salt group: injecting normal saline into tail base part of 1 week and 3 weeks, and treating by virus in 5 weeks to obtain 1 × 10 infection of each mouse 4 CFU PR8 virus, continuously recording the weight of the mice for 14 days; normal group: without treatment, the body weight of the mice was continuously recorded for 14 days.
B: after challenge, the survival rate of the mice was recorded;
c: after 4 days of virus challenge, separating lung tissues and determining virus titer;
d: after 4 days of challenge, lung tissue is separated and lung index is measured;
e: after 4 days of challenge, lung tissue was isolated and HE stained.
Detailed Description
The present invention is further illustrated by the following specific examples, and it should be noted that, for those skilled in the art, various changes and modifications can be made without departing from the principle of the present invention, and these should be construed as falling within the scope of the present invention. The experimental procedures in the following examples are conventional unless otherwise specified. The experimental materials used in the following examples were purchased from a conventional biochemical reagent store unless otherwise specified.
Materials and equipment:
(1) Gold particles were purchased from donna organisms;
(2) Polyethyleneimine (PEI) was purchased from Sigma, usa;
(3) Heptafluorobutyric anhydride (HFBA) was purchased from Sigma company, usa;
(4) The cell nucleus/cytoplasmic cell component extraction kit is purchased from Biovision corporation of America;
(5) Cy5 nucleic acid labeling kit was purchased from Mirus Bio, USA;
(6) The DNA vaccine of influenza virus Nucleoprotein (NP) is synthesized by Kyozhou-Yukiyu Biotechnology Co., ltd (eukaryotic expression vector obtained by inserting NP sequence of influenza virus PR8 into PCDNA3.1 (+);
(7) The influenza virus is a laboratory preserved virus strain PR8;
(8) 1, 2-dihexanoyl lecithin was purchased from Shanghai Allantin Biotechnology, inc.;
(9) The extruder and filter membranes were purchased from default machines (shanghai) ltd;
(10) CD3, CD4, CD8 flow antibodies were purchased from eBioscience, usa;
(11) DOPC and cholesterol were purchased from Avanti Polar Lipids, USA;
(12) FRET reagent pairs NBD-PE and Rho-PE were purchased from Biotium, USA.
Example 1
(1) VI preparation: influenza virus PR8 was ultracentrifuged and the bottom pellet was collected and 375. Mu.L of 200mM 1, 2-dihexanoyl lecithin was added to each 5mg of pellet in an ice bath for 30min. Ultracentrifugation is carried out to collect supernatant, the supernatant is placed in HBS buffer solution for dialysis for 24h, and 1, 2-dihexanoyl lecithin is removed to obtain recombinant VI, wherein the ultracentrifugation conditions are 4 ℃,100,000 Xg and 1.5h;
(2) Fluorinated modified cationic polymer synthesis: reacting according to the molar ratio of amino, heptafluorobutyric anhydride and triethylamine in the cationic polymer of 1:3:1.2, wherein the reaction medium is methanol, and stirring for 48h at room temperature. After the reaction is finished, dialyzing the reaction product in deionized water with the pH value of 3-4 for 2d, and freeze-drying to obtain a fluorinated modified cationic polymer, wherein the cationic polymer used in the embodiment is 25kDa PEI;
(3) Preparation of FAu: taking 3X 10 9 Slowly dripping gold particles with the particle size of 10-30 nm into 0.5mL of fluorinated modified cationic polymer, slowly shaking for 30min, centrifuging at 10000rpm for 10min, and collecting precipitates to obtain fluorinated particles FAu with small particle size;
(4) Preparation of ARV: and (4) mixing the FAu obtained in the step (3) with a DNA vaccine of influenza virus Nucleoprotein (NP), slowly shaking for 30min, wherein the mass ratio of the cationic polymer to the DNA is 1.5, and obtaining the DNA vaccine-loaded small-particle-size fluorinated particles (pFAU). And (2) mixing VI in the step (1) with pFAU, and extruding back and forth for 10 times by using an extruder, wherein the size of a filter membrane hole of the extruder is 100nm, and centrifuging at 10000rpm for 10min to collect the nano vaccine ARV.
And (3) taking the prepared ARV to perform characterization observation through a particle size analyzer and a transmission electron microscope. As shown in FIG. 2A, ARV size was 68.2nm, polydispersity index (PDI) was 0.21; as shown in FIG. 2B, the ARV potential was-5.6 mV; as shown in fig. 2C, the ARV is spherical in morphology.
Example 2
The low pH conditions of cellular endosomes/lysosomes induce conformational changes in influenza virus Hemagglutinin (HA), resulting in fusion of the viral envelope with the endosomal/lysosomal membrane. Therefore, when HA adsorbed on erythrocytes undergoes conformational changes under acidic conditions, it causes rupture of the erythrocyte membrane, releasing heme, and a hemolytic reaction. Thus, hemolysis experiments can be used to simulate the fusion process of membranes. As shown in FIG. 3A, ARV and VI prepared in example 1 were incubated with erythrocytes, OD 540 As the UV absorption increases with decreasing pH, both ARV and VI have hemolytic effects, indicating that membrane fusion has occurred. Further utilizing anionic liposome to simulate the membrane structure of cell, and encapsulating fluorescence energy resonance transfer (FRET) compound, which comprises the pair of NBD-PE and Rho-PE of DOPC, cholesterol and FRET reagent, wherein, the ratio of DOPC: cholesterol: NBD-PE: the mol ratio of Rho to PE is 10: 1: 0.1: 0.05. At pH5.5 and 7.4, ARV and anionic liposomes were incubated together, and the fluorescence intensity of liposomes (450 nm/595 nm) was measured at specific time points, and the lipid fusion rate = (I) t -I 0 )/(I-I 0 ) X 100%, wherein 0 Fluorescence intensity before incubation, I t For the fluorescence intensity at each assay time point, I is the fluorescence intensity of liposomes after treatment with 0.1% (v: v) Tween X-100. As shown in fig. 3B, after 1 hour of incubation, the membrane fusion rate of ARV was 35% at pH5.5 and less than 10% at pH7.4, indicating that ARV can fuse cell membranes efficiently under the acidic conditions of lysosomes. In order to examine the cell nucleus delivery capacity of the ARV on the plasmid DNA, the plasmid DNA is marked by using a Cy5 nucleic acid marking kit, the plasmid DNA is incubated with the mouse dendritic cell DC2.4 for different time, the cell nucleus is extracted, and the Cy5 fluorescence intensity of the cell nucleus is detected. As shown in FIG. 3C, the ARV nuclear fluorescence intensity was significantly enhanced (P < 0.005) compared to the ARV' group without fluorinated modified cationic polymer, and thus the ARVCan effectively deliver plasmid DNA into cell nucleus.
Example 3
Mice (C57 BL/6, 20. + -.2 g, male) were immunized with ARV prepared in example 1 by caudal subcutaneous injection at week 1, week 3 at a dose of 40. Mu.g ARV protein each. Spleen was separated at 4 weeks to prepare single cell suspension, 200 ten thousand cells were collected into 1.5mL centrifuge tubes, centrifuged at 1600rpm for 5min, the supernatant was removed and gently washed twice with 1mL PBS, finally 100. Mu.L PBS was resuspended, cells were labeled with CD3, CD8 antibody and CD3, CD4 antibody, and incubated at 4 ℃ in the dark for 30min. Subsequently, the sample was fixed with 4% paraformaldehyde and subjected to flow detection. As shown in FIGS. 4A-4B, VI, pFAU and ARV were effective in inducing CD8 in vivo + T and CD4 + T cell content increased, with ARV being most inducible. Serum samples of immunized mice were prepared at week 4 and the IgG titer in the serum was measured, as shown in FIG. 4C, the ARV prepared had a strong humoral immune effect on the mice, with significant differences (P < 0.05, P < 0.01) compared to VI and pFAU. Therefore, the nano vaccine can effectively induce in vivo humoral immunity and cellular immune response.
Example 4
20. + -.2 g of male C57BL/6 mice were selected and injected subcutaneously into the tail at week 1 and week 3 with 40. Mu.g of ARV protein per dose, prepared as described in example 1. Challenge at week 5, 1X 10 infection per mouse 4 CFU PR8 virus, followed by two consecutive weeks of weight and survival recording of mice; saline group mice were challenged by injecting Saline equal to ARV subcutaneously into caudal base at weeks 1 and 3, and each mouse was infected with 1X 10 4 CFU PR8 virus, followed by two consecutive weeks of weight and survival recording of mice; normal group mice were left untreated, and mouse body weight and survival were recorded for two consecutive weeks. As shown in FIG. 5A, the body weight of the mice in the normal group slowly increased with the lapse of time, while the body weight of the mice in the salt group continuously decreased, and the body weight of the mice in the ARV-immunized group was between the two, and decreased at 1 to 4 days, and the body weight showed a tendency of slowly increasing after 5 days. As shown in FIG. 5B, all mice in the salt group died at 12 days, whereas no death occurred in the ARV-immunized and normal groups. Lung virus dropStatistical results of the levels and pulmonary indices indicate that the lung virus content and lung damage can be significantly reduced after ARV immunization (fig. 5C-5D). The lung tissue HE staining shows that (figure 5E) a large amount of inflammatory cell infiltration appears in the lung after toxin attack, and the normal group and the ARV immune group have good cell forms, complete tissue structures and no obvious inflammation phenomenon. Therefore, the prepared nano vaccine has obvious immunoprophylaxis effect on influenza virus.

Claims (5)

1. The bionic nano vaccine coated by the influenza virosome is characterized by comprising the influenza virosome, small-particle-size fluorinated particles and a DNA vaccine, wherein the bionic nano vaccine is a nano system with a core-shell structure, the influenza virosome is a lipid vesicle containing influenza virus envelope protein, the inner core of the nano system is the small-particle-size fluorinated particles loaded with the DNA vaccine, the influenza virosome is coated on the surface of the inner core, the influenza virosome is derived from influenza virus, the influenza virus comprises H1N1, H3N2, H5N1, H7N9 or H9N2 subtypes, the small-particle-size fluorinated particles are of a nano structure formed by coating gold particles with a fluorinated and modified cationic polymer, the cationic polymer is a gene vector and comprises one or more of polyamines, polyamidoamines, hyperbranched polyamidoamines, polymethacrylates, polyaminoacids, polyesters or natural polysaccharides, and the particle size of the gold particles is 10-30 nm.
2. The method for preparing the bionic nano vaccine coated by the influenza virosomes according to claim 1, which is characterized by comprising the following steps:
1) VI preparation: adding 1, 2-dihexanoyl lecithin into influenza virus for ice bath, ultracentrifuging for 1.5h, collecting supernatant, dialyzing the supernatant in HBS buffer solution, and removing 1, 2-dihexanoyl lecithin to obtain recombinant VI;
2) Fluorinated modified cationic polymer synthesis: reacting according to the molar ratio of amino, heptafluorobutyric anhydride and triethylamine in the cationic polymer of 1.3, wherein the reaction medium is methanol, stirring at room temperature, dialyzing the reaction product in deionized water with pH of 3-4 after the reaction is finished, and freeze-drying to obtain a fluorinated modified cationic polymer;
3) Preparation of FAu: slowly dripping gold particles with the particle size of 10-30 nm into the fluorinated modified cationic polymer, slowly shaking gently, centrifuging, and collecting precipitates to obtain fluorinated particles FAu with small particle size;
4) Preparation of DNA vaccine-loaded FAu: mixing the FAu obtained in the step 3) with a DNA vaccine, and slowly shaking to obtain small-particle-size fluorinated particles loaded with the DNA vaccine, wherein the mass ratio of the cationic polymer to the DNA is 1.5 to 3;
5) Preparing a bionic nano vaccine coated by influenza virosome: and (3) mixing the VI recombined in the step (1) with the small-particle-size fluorinated particles in the step (4), extruding back and forth by using an extruder, and centrifugally collecting to obtain the bionic nano vaccine coated by the influenza virus corpuscles.
3. The method for preparing the bionic nano vaccine coated by the influenza virosomes according to claim 2, wherein the concentration of the fluorinated modified cationic polymer in the step 3) is 5 to 10mg/mL.
4. The method for preparing an influenza virosome-coated biomimetic nano-vaccine according to claim 2, wherein the DNA vaccine of step 4) comprises a full-length DNA sequence of influenza virus nucleoprotein, matrix protein, membrane protein, hemagglutinin or neuraminidase or a partial DNA sequence thereof.
5. The method for preparing the bionic nano vaccine coated by the influenza virosomes according to claim 2, wherein the size of the filter membrane hole of the extruder in the step 5) is 50-100 nm.
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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003040308A2 (en) * 2001-07-27 2003-05-15 The Government Of The United States Of America As Represented By The Secretary Of The Department Of Health And Human Services Use of sterically stabilized cationic liposomes to efficiently deliver cpg oligonucleotides in vivo
CN101827607A (en) * 2007-08-31 2010-09-08 免疫目标指定系统(Its)有限公司 Influenza antigen delivery vectors and construct
CN111848831A (en) * 2019-04-30 2020-10-30 苏州大学 Application of fluorine-containing compound modified cationic polymer in preparation of vaccine drugs

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
BR112019013402A2 (en) * 2016-12-28 2020-03-03 Invvax, Inc. INFLUENCE VACCINES

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003040308A2 (en) * 2001-07-27 2003-05-15 The Government Of The United States Of America As Represented By The Secretary Of The Department Of Health And Human Services Use of sterically stabilized cationic liposomes to efficiently deliver cpg oligonucleotides in vivo
CN101827607A (en) * 2007-08-31 2010-09-08 免疫目标指定系统(Its)有限公司 Influenza antigen delivery vectors and construct
CN111848831A (en) * 2019-04-30 2020-10-30 苏州大学 Application of fluorine-containing compound modified cationic polymer in preparation of vaccine drugs

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
Cellular gene transfer mediated by influenza virosomes with encapsulated plasmid DNA;de Jonge J, et al.;《Biochem J》;20070701;第405卷(第1期);摘要、第42页左栏第4段、右栏第2段 *
Fluorination enhances serum stability of bioreducible poly(amido amine) polyplexes and enables efficient intravenous siRNA delivery;Chen, G. et al.;《Adv. Healthcare Mater.》;20171227;第7卷(第5期);摘要、第2页左栏第2段、第12页左栏第3、4段 *
High DNA-binding affinity and gene-transfection efficacy of bioreducible cationic nanomicelles with a fluorinated core;Wang, L. H. et al.;《Angew. Chem., Int. Ed.》;20151120;第55卷;第755页右栏末段-第756页左栏首段 *
Influenza virosome/DNA vaccine complex as a new formulation to induce intra-subtypic protection against influenza virus challenge;Masoumeh Tavassoti Kheiri, et al.;《Antiviral Research》;20120715;第95卷(第3期);第2.1、2.2节 *
氟化超支化聚酰胺-胺作为流感DNA疫苗递送载体的研究;范雪莲等;《药学学报》;20200120;第55卷(第6期);全文 *

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